The influence of temperature- and divalent-cation-mediated aggregation of β-casein on the physical and microstructural properties of β-casein-stabilised emulsions

The objective of this study was to assess the influence of self-association of β-casein (β-CN) induced by both increasing temperature (5–55 °C) and divalent cation addition (Ca²⁺ or Mg²⁺) on the properties of β-CN-stabilised emulsions. The particle size of 0.5% (w/w) β-CN in 10 mM imidazole/HCl buffer (pH 6.8) was determined as a function of temperature and addition of divalent cations. Addition of CaCl₂ caused a greater increase in protein particle size than MgCl₂. Oil-in-water emulsions stabilised with 0.5% (w/w) β-CN, β-CN with added CaCl₂ or MgCl₂ (β-CN/Ca and β-CN/Mg, respectively) were also investigated as a function of temperature using light scattering, analytical centrifugation, rheology and confocal laser scanning microscopy (CLSM). Emulsions prepared with β-CN/Ca flocculated after incubation at 55 °C for 20 min and displayed significantly different physical properties (p < 0.05) compared to emulsions stabilised with β-CN or β-CN/Mg in the temperature range 5–55 °C. Based on CLSM analysis and analysis of the interfacial protein load, this flocculation was attributed to the interaction of adsorbed β-CN between droplets and the interaction of adsorbed and non-adsorbed β-CN aggregates in the aqueous phase via calcium bridges. Furthermore, the flocculation of β-CN/Ca emulsions was reversible upon cooling, which is similar to that of β-CN/Ca in solution. In conclusion, the temperature-dependent behaviour of β-CN-stabilised emulsions correlated to the temperature-induced aggregation of β-CN, particularly in the presence of Ca²⁺. Hence, the stability of β-CN-stabilised emulsions can be predicted from the extent of β-CN aggregation in aqueous solution (i.e., aggregate size).